Loratidine is associated with improved prognosis and exerts antineoplastic effects via apoptotic and pyroptotic crosstalk in lung cancer

Background Tumor-associated inflammation suggests that anti-inflammatory medication could be beneficial in cancer therapy. Loratadine, an antihistamine, has demonstrated improved survival in certain cancers. However, the anticancer mechanisms of loratadine in lung cancer remain unclear. Objective This study investigates the anticancer mechanisms of loratadine in lung cancer. Methods A retrospective cohort of 4,522 lung cancer patients from 2006 to 2018 was analyzed to identify noncancer drug exposures associated with prognosis. Cellular experiments, animal models, and RNA-seq data analysis were employed to validate the findings and explore the antitumor effects of loratadine. Results This retrospective study revealed a positive association between loratadine administration and ameliorated survival outcomes in lung cancer patients, exhibiting dose dependency. Rigorous in vitro and in vivo assays demonstrated that apoptosis induction and epithelial-mesenchymal transition (EMT) reduction were stimulated by moderate loratadine concentrations, whereas pyroptosis was triggered by elevated dosages. Intriguingly, loratadine was found to augment PPARγ levels, which acted as a gasdermin D transcription promoter and caspase-8 activation enhancer. Consequently, loratadine might incite a sophisticated interplay between apoptosis and pyroptosis, facilitated by the pivotal role of caspase-8. Conclusion Loratadine use is linked to enhanced survival in lung cancer patients, potentially due to its role in modulating the interplay between apoptosis and pyroptosis via caspase-8. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-023-02914-8.


Introduction
Lung cancer, with an incidence rate of 11.4% and a mortality rate of 18%, significantly threatens human survival and social development [1].The complex relationship between inflammation and tumor progression suggests that inflammatory microenvironments could either prevent or promote tumor formation.On the one hand, chronic inflammation shares similarities with cancer, such as increased angiogenesis and apoptosis inhibition [2].On the other hand, it may prevent tumors by enhancing immune surveillance resulting from an immune response [3,4].Studies have shown that antiinflammatory and antioxidant treatments may prevent or delay cancer onset and development.Nonsteroidal anti-inflammatory drugs, such as aspirin, metformin, and statins, have anticancer effects, along with other cell and inflammatory factor inhibitors [5,6].Recent research has begun to explore the potential of antihistamines in cancer treatment.
Antihistamines, functioning as antagonists or inverse agonists of histamine receptors, are employed in the management of conditions such as allergic rhinitis, allergic conjunctivitis, and urticaria [7].Recent investigations have posited a potential application of antihistamines such as cimetidine, terfenadine and astemizole in the realm of cancer therapy [8][9][10][11].For instance, some researchers observed that histamine can prevent the proliferation of a diverse array of cancerous cells, suggesting that antihistamines may serve as viable inhibitors of tumor expansion [12].Shah and Stonier proposed that the influence of these drugs on cellular growth, apoptosis, and angiogenesis may confer anticancer properties [13].Additional researchers have documented associations between antihistamine utilization and decreased risk of colorectal, breast, and lung cancers, underscoring the need for further exploration of the potential advantages of antihistamines in cancer prevention and management [14,15].
The aforementioned studies offer an expanded perspective on the possible implications of antihistamines in cancer therapy.However, their exact therapeutic potential in Asian people and the most effective approaches for incorporating them into cancer treatment regimens remain to be clarified.As a result, our study aims to examine the anticancer mechanisms of loratadine, a commonly employed antihistamine, by employing Cox regression models to identify potential associations between noncancer drug exposure and patient prognosis.To corroborate our findings from the retrospective analysis and investigate the antitumor efficacy of the candidate agent, we conducted observations utilizing cultured cells, various animal models, and computational assessments of RNA-seq data.
The concomitant use of loratadine and other medications exhibited a reduced mortality risk compared to the administration of these drugs individually (Fig. E1).A dose-dependent relationship was observed between cumulative loratadine consumption and a decline in lung cancer mortality (p = 0.015 for trend test, Fig. 1A).Additionally, significant disparities were detected in the OS and PFS curves when comparing patients with and without loratadine treatment (Fig. 1C-D).
Taking into account the impact of chemotherapy on drug utilization, lung cancer risk, and mortality, the magnitude of the association between drug use and OS or DFS exhibited minor variations in the stratified analysis based on chemotherapy.Nevertheless, the direction of the association remained consistent with the overarching findings.In light of potential toxic side effects, loratadine was chosen as the drug of focus for this investigation.

Moderate doses of loratadine may induce cell senescence and apoptosis while inhibiting epithelial-mesenchymal transition (EMT)
Loratadine was tested to discover the potential therapeutic mechanism in vitro and in vivo.First, the IC50 values of lung cell lines were calculated according to the results of CCK8 assays (Fig. E2).
For in-depth analysis, we treated A549 cells with moderate loratadine concentrations (IC50) or DMSO (as a control to exclude effects of the solvent) for 48 h and then performed RNA sequencing (RNAseq) analysis.Principal component analysis (PCA), volcano plot analysis and heatmap of RNA-sequencing data separated loratadine-treated from control groups (Fig. 2A-C).Critically, enrichment analysis performed on the differentially expressed genes revealed many overlapping processes, particularly those related to cell senescence and negative regulation of the vascular endothelial cell proliferation signaling pathway (Fig. 2D and E, Figs.E3 and E4).
To gain insight into the mechanism, we built a loratadine-targeted gene set based on the CTD mentioned above, of which genes were differentially expressed in the two groups (Fig. E5A).Subsequently, GO and KEGG enrichment analyses based on loratadine-targeted gene expression were further explored (Fig. E5B-G).Interestingly, the cell senescence signaling pathway was also enriched.In addition, pathways including the cell cycle, P53 signaling pathway and apoptosis were identified.Among them, cell senescence and apoptosis are frequently prevented by epithelial-mesenchymal transition (EMT) [16], which is known to broadly regulate cancer invasion and metastasis.As active caspase-3 and caspase-8 have been reported to enhance apoptosis, we therefore assessed whether loratadine induced apoptosis by evaluating caspase-3/8 activity and BLC-2 reduction.Western blot analysis revealed increased cleavage of caspase-3, caspase-8, Bax, and PARP, while Bcl-2 levels decreased in loratadine-treated cells compared to the control group (Fig. 3G and H).Moreover, we carried out a scratch wound assay and live cell imaging of A549 (Fig. 2F, movies 1 and 2), PC9 (Fig. 2I, movies 3 and 4), and HCC827 cells (Fig. 2J, movies 5 and 6) as a functional test for cell migration and cell status in vitro.As indicated in Fig. 2G, drug treatment notably reduced the migratory ability of A549 cells in the 72-h group.The average level in the loratadine group was lower than that in the control group, with a difference of -0.124 (-0.221--0.026),and the difference was statistically significant (t = -3.247,P = 0.023).Loratadine treatment also impacted colony formation, resulting in an 80-90% reduction compared to the control group (Fig. 2H).

High-dose loratadine promotes pyroptosis and interacts with apoptosis
Apoptotic signaling, which involves the activation of caspases, such as caspase-8 and caspase-3, can induce other types of cell death.Therefore, we administered a high dose of loratadine (twofold IC50, H-loratadine) to further investigate its effects.After 48 h of treatment, compared with the DMSO group, cells in the loratadine    group displayed distinct membrane blebbing morphological changes when observed under a microscope (Fig. 3A).Colony formation with H-loratadine resulted in a variable effect, with over a 90% reduction in colony formation compared to the DMSO group (Fig. 3B).Flow cytometry analysis revealed that high doses of loratadine can induce pyroptosis, with a significantly increased proportion of Annexin V and PI double-positive staining in the high-dose loratadine group (70.667 ± 2.558%) compared to the control group (9.773 ± 1.315%), and the difference was statistically significant (P < 0.05) (Fig. 3C-F).The cleavage and activation of GSDMD or GSDME by inflammatory caspases, such as caspase-1, caspase-3, caspase-4 and caspase-8, can be assessed in the H-loratadine group by Western blot analysis.(Figs.3J, I and  4E).During our investigation, we made a novel discovery that H-loratadine may stimulate the upregulation of PPARγ expression (Fig. 4F), which in turn acts as a promoter of GSDMD, as verified through a luciferase reporter assay (Fig. 4B).Using ELISA to detect the concentration of IL-18 in the cell supernatant, it was found that the high loratadine group was linked to an elevated release of proinflammatory cytokines, and the difference was statistically significant (P < 0.001) (Fig. 3L).

Loratadine treatment suppresses tumorigenesis and activates p53 and GSDMD in C57BL/6 mice
Piqued by the clinical and ex vivo findings, we sought to determine whether loratadine exhibits tumor-suppressive properties.Lewis cells (LLCs) were implanted in the right flank region of C57BL/6 mice, and metastatic colonization capacity was evaluated by assessing tumor nodule formation in the lungs after LLC tail vein injection.All models were administered loratadine or vehicle via intragastric administration.Tumor size measurements demonstrated that loratadine diminished tumor cell growth and metastasis in C57BL/6 mice in a dose-dependent manner (Fig. 5A-D).Moreover, the vascular morphology of mouse tumor sections was stained with DAPI and CD31 to evaluate the antivascularization ability of loratadine.Although the loratadine-treated group exhibited reduced microvessel density compared to vehicle-treated mice, this difference was not statistically significant in C57BL/6 mice (Fig. 5E-G).IHC staining verified a minor decrease in the levels of Ki67 with a significant increase in p53 and GSDMD staining in loratadine-treated LLC tumors (Fig. 5F and H-J).In conclusion, these studies indicate that loratadine may prevent tumors from growing, presumably through the regulation of proliferation via apoptotic and pyroptotic pathways as well as the regulation of the immune system.

Discussion
A key finding of our study is that lung cancer patients receiving loratadine exhibit improved survival outcomes.Significant differences were observed in OS curves and PFS curves between patients with and without loratadine use.As the loratadine dose increased, patients experienced a significant improvement in outcomes.The results from animal experiments also conformed to the clinical outcomes, suggesting dose-dependent improvement.Our findings not only align with but also expand upon previous research.A study examining the impact of antihistamine use among Danish patients found that loratadine use was significantly linked to reduced allcause mortality in patients with NSCLC or any cancer when compared to the usage of antihistamines that are not CAD [15].Another nationwide Danish cohort also demonstrated that the use of antihistamines was related to a prognostic benefit in patients with ovarian cancer [17].Furthermore, a recent study reported an association between improved survival in melanoma and breast cancer patients and the use of H1-antihistamines desloratadine and loratadine [18].Recent retrospective analyses have shown that cancer patients who received antihistamines during immunotherapy treatment demonstrated dramatically enhanced survival outcomes [19].
M-loratadine have been found to influence a range of cellular pathways, notably those pertaining to the the cell cycle, cell senescence, P53 signaling pathway and apoptosis.Empirical evidence from scratch wound assays, colony formation assays, and Western blot analyses confirms that M-loratadine can concurrently induce cell senescence and apoptosis in addition to inhibiting epithelial-mesenchymal transition (EMT).This observation is in alignment with previous findings that suggest cell senescence and apoptosis may be hindered by the EMT process-a phenomenon that could enhance cellular survival during EMT [16].Recent literature further substantiates a complex interconnection between apoptosis and senescence, indicating that both processes may be regulated by shared mitochondrial-dependent pathways [20].In addition, these findings align with previous studies suggesting loratadine's antitumor effects through inhibiting the cell cycle or inducing "silent" cell death, such as apoptosis.For instance, loratadine may result in G2/M phase cell cycle arrest and death in colon cancer cells (COLO 205) [21].Consistently, loratadine reverted multidrug resistance in NSCLC, breast and prostate cancer cells and sensitizes NSCLC cells to chemotherapy by inducing apoptotic and lysosomal cell death [15].Loratadine may also directly damage DNA and activate Chk1, promoting G2/M arrest and making cells more susceptible to radiation-induced DNA damage while downregulating total Chk1 and Cyclin B [22].Besides, researches on loratadine combination therapy also proved effective in cancer treatment.Loratadine, in combination with thioridazine may inhibit the rapamycin signaling pathway via phosphoinositide 3-kinase/ Akt/mammalian target in gastrointestinal tumor [23] and effectively overcome immune evasion by suppressing CRC growth in a mouse model [24].Moreover, H1-antihistamine treatment can enhance immunotherapy response via activation of the macrophage histamine receptor H1 [19].
Our study has uncovered some intriguing and novel findings.High-dose loratadine induced pyroptosis, a new form of cell death, alongside traditional apoptosis.Detailed analysis of cell models treated with high doses of loratadine and various inhibitors suggests a significant role for caspases in mediating both apoptotic and pyroptotic pathways.A critical discovery was that high-dose loratadine augmented the PPARγ level, which subsequently spurred GSDMD transcription by our luciferase reporter assay.This finding is further substantiated by reports in the literature indicating that PPARγ is instrumental in the regulation of caspase-8 activation [25].Tumor sections from animal models, stained for Ki67 and GSDMD, reinforce the hypothesis that loratadine hampers tumor proliferation by promoting pyroptosis.Collectively, these results highlight a novel mechanism by which loratadine may exert anti-tumoral effects through the induction of pyroptosis.
The interplay between pyroptosis and apoptosis is intricate.They are two distinct forms of programmed cell death that are crucial for maintaining cellular homeostasis and responding to varying loratadine dosages.Caspase-8, a critical molecule that interconnects apoptosis and pyroptosis, can promote apoptosis via the extrinsic pathway and regulate pyroptosis by interacting with inflammasomes and modulating inflammatory caspases and gasdermin D (GSDMD) or gasdermin E (GSDME) activity by activating other caspases, such as caspase-1, caspase-4/5, and caspase-3 [26][27][28][29].Various studies have emphasized the crosstalk between apoptosis and pyroptosis in different contexts.Orning et al. demonstrated that caspase-8 acts as a regulator of GSDMD-driven cell death and can cleave GSDMD during bacterial infection by Yersinia, leading to pyroptosis and the release of proinflammatory cytokines [26].Sarhan et al. demonstrated that activating caspase-8 leads to the cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in macrophages, which results in pyroptosis.The absence of GsdmD causes a delay in membrane rupture, which in turn prevents the morphological changes associated with cell death from transitioning to apoptosis.This finding highlights caspase-8's dual role in modulating both apoptosis and pyroptosis, depending on the cellular context [27].These studies underscore the importance of understanding the interplay between apoptosis and pyroptosis for cancer treatment, as targeting this crosstalk may provide new therapeutic opportunities for treating various cancers.However, our investigation had certain limitations.First, the retrospective and single-center nature of the study might introduce unidentified biases that could affect our analysis.Additionally, our study did not sufficiently investigate the greater detail in crosstalk between apoptosis and pyroptosis.In summary, we utilized a large case-control population, virtually complete cancer ascertainment and histological verification, and detailed prescription data.The continuous updating of prescription data, along with accurate information on drug type and quantity, allowed for an extensive evaluation of exposure patterns and eliminated recall bias.
Mechanistically, loratadine has the potential to incite a complex interplay between apoptosis and pyroptosis mediated by the crucial role of caspase-8.This is accomplished via the modulation of PPARγ levels that in turn stimulate gsdmd transcription and activate caspase-8 (summarized in Fig. 6).Further studies on any effects of other antihistamines or their immune response to tumors may also be merited.

Translational relevance
The identification of loratadine's anticancer mechanisms in lung cancer, including its dose-dependent effects on apoptosis, EMT, and pyroptosis, along with its ability to augment PPARγ levels and enhance caspase-8 activation, holds significant translational relevance.These findings suggest the potential of repurposing loratadine as an adjuvant therapy in lung cancer treatment.By modulating the interplay between apoptosis and pyroptosis, loratadine offers a promising avenue for improving patient outcomes.Further research could explore the clinical application of loratadine in combination with existing treatments and investigate its efficacy in other cancer types.Overall, this study provides valuable insights that may expand the therapeutic options and contribute to the development of novel strategies for lung cancer patients, addressing an unmet clinical need.• thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Fig. 1
Fig. 1 Uptake of loratadine is correlated with better survival in patients diagnosed with lung cancer.A Lung cancer mortality decreased in a dose-dependent manner with the increasing cumulative use of loratadine.B Hazard ratio for different drugs in the univariate analysis of OS.C-D The overall survival curve and disease-free survival curve with and without loratadine use

Fig. 2
Fig. 2 RNA sequencing of loratadine treatment in vitro.A PCA of RNA sequencing samples.B A549 cells were activated with loratadine (IC50) for 2 days (n = 3 biologically independent samples per group).RNA sequencing was performed.Differential gene expression is shown in a volcano plot.C A549 cells were activated with loratadine (IC50) for 2 days (n = 3 biologically independent samples per group).RNA sequencing was performed.Differential gene expression is shown in the heatmap.D GO enrichment categories of DEGs.E KEGG enrichment categories of DEGs.F Wound healing assay and live cell imaging of A549 cells.G Each column represents the mean value of the migrated area of the two different groups in A549 cells, and error bars indicate SD. *, p < 0.05.H Cell growth was examined by colony formation assays in various lung cancer cell lines.I Wound healing assay and live cell imaging of PC9 cells.J Wound healing assay and live cell imaging of HCC827 cells

Fig. 3
Fig. 3 High-dose loratadine promoted the crosstalk between apoptosis and pyroptosis.A Evident balloon-like morphological changes were noted with red arrows in different cell lines treated with H-loratadine.B Cell growth was examined by colony formation in lung cancer cell lines treated with H-loratadine and combined with a GSDMD inhibitor.C to F Flow cytometry analysis of inhibitor-treated A549 cells stained with Annexin V-FITC and PI.The percentage of double-positive cells was presumably pyroptotic cells.G-K A549, PC9, and NCI-H1299 cells were treated with different doses of loratadine.The indicated proteins were analyzed by Western blotting.Dose-dependent cleavage of caspases, p53, p21, bax, PARP, GSDMD and GSDME was demonstrated.GSDMD-FL, full-length GSDMD; GSDMD-N, GSDMD N-terminal domain.GSDME-FL, full-length GSDME; GSDME-N, GSDME N-terminal domain.L Release from A549 cells treated with H-loratadine.Each column represents the mean value of three biological replicates, and error bars indicate SD. ***p < 0.001

Fig. 4
Fig. 4 Pharmacological agents and caspase inhibitors partially reversed loratadine-induced cell death.A Pyroptotic cell changes, such as cell swelling and membrane blebbing, were noted with red arrows in A549 cells treated with H-loratadine as well as different cell death inhibitors.B Validation of PPARG as a promoter of GSDMD expression using a luciferase reporter assay.The promoter region of GSDMD was cloned upstream of the luciferase gene in the pGL3-Basic vector.The PPARG expression vector (vec PPARγ) was used to upregulate PPARγ expression.Bar graph representing relative luciferase activity in cells.Luciferase activity was normalized to Renilla luciferase activity for each sample.Data are presented as the mean ± SD of three independent experiments.***P < 0.001 compared to control.C-D A549 and NCI-H1299 cells were treated with different doses of loratadine combined with cell death inhibitors or caspase inhibitors.The indicated proteins were analyzed by Western blotting.GSDME-FL, full-length GSDME; GSDME-N, GSDME N-terminal domain; C1 IN, caspase 1 inhibitor (ac-YVAD-cmk); C3 IN, caspase 3 inhibitor (z-DEVD-fmk); C8 IN, caspase 8 inhibitor (z-IETD-fmk); Z-VAD pan caspase inhibitor (z-VAD-fmk); M-LOR, moderate dose of loratadine; H-LOR, high dose of loratadine

Fig. 5
Fig. 5 Loratadine treatment suppresses tumorigenesis in vivo.A Schematic overview of the mouse experimental model protocol.B Micro-CT images of lungs from mice following LLC tail vain injection with or without loratadine treatment.Three-dimensional rendering of micro-CT data with lungs in gray; the lost part represents the tumor.C-D The indicated cells were subcutaneously injected (0.5-1 × 10. 6 cells per mouse) into C57BL/6 mice (n = 5).Tumor formation was analyzed.CON, vehicle group; L1, 5 mg/kg/d resveratrol group; L2, 35 mg/kg/d resveratrol group; and L3, 175 mg/kg/d resveratrol group.After 7 days of treatment, 4 mice died in the L3 group.D Vascular morphology of mouse subcutaneous tumor sections stained with DAPI (blue) and CD31 (red).Magnification: IHC 10 × and 20 × , Scale bar: 500 µm and 200 µm.E Tumor sections stained with p53, Ki67 and GSDMD.Magnification: IHC 20 × and 40 × , Scale bar: 200 µm and 100 µm.F Each violin plot represents the quantification of vascular density.ns, no significant difference.G-I Each violin plot represents the relative expression levels of each tumor section protein.Data are presented as the means ± SDs. ns, no significant difference, **p < 0.01, ***p < 0.001 (See figure on next page.)

Table 1
Clinicopathological characteristics of patients

Table 2
Hazard ratio for univariate analyses of DFS in different drugs